Science Fireside Chat (Q & A): [2018 Conference Video]

This blog is based on a presentation at the FPWR 2018 conference. You can watch the complete presentation by clicking on the embedded video. In case you don't have time to watch the full video, we've included a full transcript below. You can also watch the full set of conference videos on YouTube.

Video Transcript

Theresa Strong:

Thank you everybody for joining us this afternoon, I am thrilled that we have three Molecular Genetics/Cellular Biology scientists with us today for all of you to chat with. I think our goal in this session was to give you an opportunity to learn a little bit about what each of our scientists are doing and also have the opportunity to ask some questions and have them answered. We're thrilled to have Larry Reiter from University of Tennessee Health Sciences Center, Rachel Wevrick from the University of Alberta, and Rob Nichols from the Pittsburgh University Children's Hospital of Pittsburgh. I'm going to give them each a couple of minutes to introduce themselves and the kind of work that they work on and then we're going to take questions. The idea here is it is fair game to ask almost anything; about their opinions. What we hope to see is the most interesting things in the field, the promising areas of research, and also if you have questions about the research that is going on right now. I will let Rob lead off.

Rob Nichols:

I'm Rob Nichols. I started work on Prader-Willi syndrome in 1987 (a long time ago) and at that point there were no genes known. Imprinting wasn't known. I found that during my postdoctoral work that genomic imprinting played a role with Prader-Willi syndrome, so I continued to work on PWS for my career; the first decade or so doing human genetics and then switched for another 10 or 12 years to working on mouse models. We characterized a deletion mouse model of PWS and failure to thrive. As a consequence of hypoglycemia, the mouse only lived a week. We characterized the cause and it was actually associated with pancreatic hormone defects of defects in secretion of insulin and glucagon. We're currently working on a cellular model. We've generated PWS pancreatic beta-cells and looking at the mechanisms by which insulin is secreted. That's one ongoing area that started from the mouse but we're now using cell models for that. Then about a handful of years ago decided that because mouse models were developing hyperphagia obesity, because they were surviving, that we wanted to generate a different animal model. I started working on trying to create a pig model (mini-pig) and so I first had to do the genomics and then collaborated with a pig group in Missouri to establish a pig PWS model. We've had the pigs in Pittsburgh since May and now we're breeding. It’s been quite an experience just diving in and starting to work with pig mothers. None of us had ever had any experience with pigs before. They're like having dogs. You know there's always things going on.

TS:

Just for background; the pigs offer some advantages as far as the physiology is more like humans and they have more complex behaviors.

RN:

It turns out that the brain development in pigs is much more like humans. The brain development, the brain size, the structure of the brain, and the highly folded parts of the brain that are associated with intelligence—that's more developed. For example, the pig is even lower in primates like monkeys. To get closer to a human you basically have to go to either chimpanzees or gorillas. For a combination of ethical and costing reasons, it's difficult to work with great apes. We hope that the pig is going to be an excellent model. The disadvantage is that there's a much longer lifecycle. It’s a three month pregnancy and then it's seven months to maturity before you can breed. It’s basically a year per generation and we don't have the ability to do genetics like with the mouse model, in terms of more complexity and greater cost. They are different systems, but we hope that the pig model develops with the genetic mutation and these pigs have the imprinting defect mutation. We're hoping that they provide a really powerful resource.

Rachel Wevrick:

Rachel Wevrick. I’m at the University of Alberta in Edmonton, Alberta, Canada. I first became interested in Prader-Willi syndrome, working in a lab at Stanford University as a postdoctoral fellow and then moved to the University of Alberta in 1996. I had been looking at first, the genetics of Prader-Willi syndrome, and identifying some of the genes that are within the deletion region (the ipw gene, necdin gene, MAGEL2 gene). You've probably heard some of these strange name genes coming through in some of the talks. Then we started looking at the effect of loss of those genes in mouse models. We looked at mice that were missing the necdin gene, that have deficiencies in muscle and brain formation, as well as in fact tissue. Then we looked at deficiencies in the MAGEL2 gene and those mice also have deficiencies in in the hypothalamus; specifically, in endocrine function. After that we started transitioning more to two programs of research; one more in the neurobiology of leptin deficiency and MAGEL2 deficiency. The idea behind that whole scheme is that when you have a deletion of the entire region, then it is very complicated to model because there's so many genes that contribute to the disorder. Looking at a disorder in humans or complementary disorder in mice, when you're looking at several genes at the same time, the idea was that you could break that down and look at just one gene at a time then gather the effects of all of those genes (in some sense you could look at the whole sum of the parts). We started looking more at the neurobiology of both the MAGEL2, necdin genes, and the proteins that are encoded by those genes; then the proteins that those proteins associate with. We have the MAGEL2 protein and what we wanted to know is what are the other proteins that are near to the MAGEL2 protein in the cell? By getting to know those neighbors we can get to know what processes the protein is involved in. Then you start to have some targets for how you can change those processes to benefit the mice (and obviously to benefit people who are missing that MAGEL 2 gene or who have mutations in the MAGEL2 gene as is the case in Schaaf-Yang syndrome that some of you may be familiar with). That neurobiology and molecular side was one avenue that we're still working on. The second avenue is looking much more directly at therapeutics that are in either in clinical trials in Prader-Willi syndrome or where companies have approached me and said, “we're considering this line of research. We'd like to look at the melanocortin system. There's good evidence that the melanocortin system is disturbed in Prader-Willi syndrome and may be responsible for some of the excessive appetite that's seen. We could really use extra evidence that there is actually a deficiency in the melanocortin system and can we test our compound in your mouse model? We've done that for Setmelanotide with development for use in a number of genetic disorders that include obesity. We looked at Diazoxide as well. You may have heard about the Diazoxide trials that are now going into phase 3 (or have results in phase 3). Both Setmelanotide and Diazoxide had very beneficial effects in this mouse model of Prader-Willi syndrome where the MAGEL2 gene is gone. With diazoxide, we saw a reduction in fat mass that's elevated in those mice and with Setmelanotide we saw a very large reduction in appetite in acute food intake in the mice. Of course, the benefit of working in animals is that you can raise large cohorts that are all genetically identical, that can all be tested at the same time, and you can also look at the end of the study at what the consequences are to the animal and to the brain. Obviously, that's not approachable in human subjects, so if doing animal research is not unique to Prader-Willi syndrome, it's extensively used in the pharmaceutical industry and extensively used in in research. That's really the rule that for most genetic disorders; there's animal research going on. Those mice were also used for a trial of a peripherally acting endocannabinoid receptor antagonist; looking at the munchie system but trying to get the anti-munchies by using the cannabinoid (cannabis system) to address appetite. There was a study of the mice that we developed and this endocannabinoid receptor antagonist. We’ve also collaborated on a study on the same mice and the MAGEL2 mice, looking at the effective vertical sleeve gastrectomy, which is stomach reduction surgery; bariatric surgery, which also had a beneficial effect on fat mass and activity in the mice. We did not do the tiny surgeries on tiny mice in my lab. They are bigger than flies. That’s true that they're bigger than flies. Those studies have been moving along as well so that's our preclinical development side and then the neurobiology and molecular cellular side to our program of research.

Larry Reiter:

I’m Larry Reiter and I think I’m going to tell you generally how I got into Prader-Willi research. I've actually known Rob Nicholls since I was graduate student and I've started working on Angelman syndrome and 15q duplication syndrome as a postdoc. My lab was ramping up and we've been working on 15q for a very long time. It really made a lot of logical sense for our next step to work on Prader-Willi syndrome. We sort of understood the genes in the region and we have some systems in place. You might know me more as the “tooth fairy guy”. I've been taking teeth and making stem cells. There are stem cells in the teeth, so we make them into neurons in a dish and then we can do lots of different studies. We've collaborated with several investigators here and we have our own research which is focused more on the autism aspects of Prader-Willi syndrome UPD. You maybe have heard that there's higher autism in kids with UPD. That was the scientific presentation I did yesterday. What we're trying to do is collect as many individuals as possible because when you do basic research you know one of the limiting factors in doing human genetics is that everybody's a little bit different; sometimes a lot different. What you need is as many biological replicates as possible. It’s very difficult to obtain those. If you're going to make things called induced pluripotent stem cells (you might have heard of)—we took the approach of making these dental pulps themselves. You guys can give us teeth. The teeth fall out and you put it in a tube and send it to the lab. We grab the stem cells. It’s simple enough that we have over 200 stem cell lines in the lab for 13 different neurogenetic disorders. We've been pretty successful with that approach thanks to the families. It’s really important that you guys continue to collect these teeth for us. We use them. We need them. It really is essential to our research. The other side of my lab I'm just going to briefly mention is that we work in fruit fly models, not mouse models. There are some serious advantages to that which we could discuss but we don't currently have any Prader-Willi models. In flies, our models are focused on the seizures that occur in 15q duplication syndrome. That's our current fly model that we've been using to actually screen for anti-seizure compounds. Other than those two things I really was hoping to field questions because I have publications and all kinds of stuff that you could look up online. You don't really want to do that you want to ask about science right?

TS:

All right. Great. Thank you. We’ll have questions from the audience but there were a few questions that were submitted through the app and I thought it would be good to kick off with one of them which is for each of you. What is one exciting finding in your area of research that's come out in the last year that you think might be interesting to people? If your research is the most exciting (which it often is) or if there's something that is related to PWS; do you have some ideas?

LR:

The most exciting thing I work on is what I just presented because it always has to be so. My concern was from years of being around Prader-Willi syndrome and working on autism and other syndromes was that I never really saw anybody looking for genes associated with Prader-Willi syndrome. I'm very excited that we were able to finally do some RNA seq (look at gene expression in Prader-Willi UPD plus and minus ASD versus deletion versus control) and that data we presented recently. There's clearly a signature there. There's lots of genes that we found there are also some known autism associated genes in that set that we found. This is very early. We got the data about a month ago so we're not close to publication really but we're excited that we actually found something and it's unique to the plus autism group. It may be trying to figure out not only that mechanism of how those genes cause autism in Prader-Willi syndrome but how they cause autism in general (and maybe in other cases of autism where that combination of gene expression is present)? So that to me is very exciting.

RW:

I think what I'm most excited about is seeing how many people see Prader-Willi syndrome as a tractable problem. I think for many years the consensus seemed to be that this was an unusual disorder, nobody was ever going to understand it because it was completely unique and used pathways that were not related to any other pathways. It’s going to be a really difficult problem. I think that between the clinical people, the preclinical people, the molecular people, and a lot of scientists who were real experts in neurobiology who've been attracted to this problem; they see it is completely tractable. They see it as we have to figure out the pathways and then each of those pathways with a compound that has worked elsewhere can work in this disorder. By breaking it down into its components I think I think we were easily up to the challenge of addressing many of the of the pathways that are disrupted.

RN:

I can follow on from Rachel to that. It has been very exciting to see many new researchers coming to the field, pharmaceutical companies that are willing to do drug trials, and the new technologies that are available in in science. We've had genome sequences that opens up not only studying of different humans but different animal models. That then allows us to utilize genetic approaches. Now we can make any mutation pretty much that we want to catch, develop new models, and we can also test therapeutic approaches using gene therapy type approaches. That's probably not the long-term way to go with Prader-Willi syndrome, but it opens up a lot of increases our ability to understand what's going on. Other technologies, as Larry mentioned his dental pulp from stem cells were potent stem cells. This has opened up the ability to generate PWS neurons and culture. It's not exactly the same as in vivo in an individual or an animal model. It presents ever better models and by utilizing all these new approaches and new investigators we're now really starting to get at the point (not just what each of the PWS genes do because it's not one, it's a dozen genes). It’s infinitely more complex than a lot of the single gene disorders; which are still complex and difficult to develop therapies. We're now getting at what these genes; do what their pathways are; as Rachel discussed what they're interacting with. By understanding the pathways, the hope is now that we can give our ideally small molecules that will specifically target the defect so that if there's specific targets then they're not going to have a wide range of target effects or side effects that you don't want. That's the exciting part now having been incredibly researched for so long. It was just a matter of slogging away and trying to even get a basic understanding. We can now see that we're going to understand the real possibility of therapies.

TS:

I'll just add to that because I spent 20 years working in the area of gene therapy (and it was a long 20 years) but gene therapy as a field is really now starting to have an impact on patients and on many different genetic disorders. Now that said, PWS is more complex. It's not a single gene where we put one gene back and it's straightforward; just the fact that all of that technology is advancing so rapidly. I was at the NIH Rare Disease Day and they've set a goal of using CRISPR technology to treat rare genetic diseases (hundreds of them). They’re trying to scale up over a 10-year period. They can have sort of an off-the-shelf crisper, technology again. It’s not perfect for PWS because that is really the technology as it is today: it's really adapted for one gene, one mutation that you can just fix. We have several genes. I still don't know exactly which one does what, but I think the overall point is that the technology is advancing. Everything is being scaled up and all of that even though it may not be easily applied to PWS; it will advance the field where we can start to tackle more complex things like PWS.

RN:

The other aspect of course, is that gene therapy is easier when you have access to the relevant tissue. The blood cells are quite relatively straightforward even for high diseases. It's being used successfully for muscle tissues. It’s not as great a risk as dealing with the brain. There are possibilities; understanding the pathways and hopefully developing small molecules or other types of things. You can consider a hormone like growth hormone or other hormones we're working on now as small molecules essentially replacing what's missing or altering the function of pathways.

TS:

Right. I think it’s good that we have both options. At the minimum, the gene therapy technology can be used to help you guys understand PWS.

Participant:

I understand that gene therapy for the replacement is all there but there's the maternal switch for the deletion patients. What do you think the future is there? How can we support that research and actually get some animal models or IPS cells that can look at the maternal switch problem?

LR:

Do you know anything about Angelman Therapeutics? They are using these things called SOS now and it's working very well. As Rob said, you have to get it where it needs to be and that's the key. The problem is actually getting it where it needs to be in the brain. Even for the SOS it's very difficult to get them into all the neurons that need to be in in the brain, right? Theoretically we could mess with the imprint; the switch that you're talking about. We still have a delivery issue to get it there; every neuron where it needs to be. If you look at the Angelman field, which in some ways is a little more advanced in terms of following when the syndrome develops in the mouse models, there's been a lot of work lately showing that when you rescue ub3 (which is the gene that I work on a lot) it makes a difference. It turns out (and a very recent paper shows) that you can rescue some of the brain function; things where you can measure electrophysiology; you can measure brain activity; you can actually rescue that and not rescue the behavior in that mouse. If you turn on ub3 in an adult but if you rescue ub3 earlier in development, you can rescue the behavior. That was surprising. I don't think people expected that, but those kinds of studies should certainly be done in Prader-Willi. There's a lot of potential there and it's a parallel track.

RN:

One of the issues of course is that the mouse brain is very small. It's 3,000 times smaller than the human brain. When you apply ASOS and there's enough diffusion of the small molecules and they hit enough neurons you can get an effect. That's a lot harder with an organ that's 3,000 times smaller, so that's where an animal model like a pig may be important. The pig brain is only 3 times smaller. It’s the same order of magnitude as a human brain. So now, if you try your ASOS or other compounds you can actually directly look at how many neurons you are now able to get to and what the effect is both in vivo in the animal model; then as necessary, post mortem to look at the effects. I think that’s good news.

TS:

The feasability questions are pretty clear. We some good models now. You have the mouse, we have the pig, we have some molecules that will turn the genes on. We can start to look at these questions around when would it have to be done? To what level? What genes have to be activated to have an effect? We are in a position at least to begin to ask those questions and look at whether this is a feasible approach for PWS. We want to do right?: What's feasible? What's not feasible? We kind of sort through all of that and pick out the things that are the most promising.

Participant:

Rob, just wondering about the phases in the pig? Does it go through the failure to thrive into the hyperphagia into the obesity? Does it follow those phases and also to second to that; is all the RNA available in the pig the same deletion?

RN:

I'll answer the second part first because that's the simplest. The answer is yes. The whole region is conserved. The region is conserved in all mammals so even in mice, rats, and pigs. In all the species all the major components are present. Then, the first question: do we have all the same clinical progression? I can only partially answer that because right now we have our founder animals and we're in the process of initiating breeding to generate experimental cohorts. Then we'll be able to answer the question much more definitively when we have a larger number of animals. Right now, we have (it sounds a little weird) 1.5 pigs with Prader-Willi. The reason we have 1.5 is because we have one pig that's full Prader-Willi with a full genetic effect we have half because he's actually a mosaic; the way that you generate using genome editing these animals. He actually has two cell types, so half his cells have the full PWS genetic effect and the other half of his cells are functionally normal (although they carry one of the mutations and the maternal chromosome sets). Both animals as piglets had hypoglycemia, which would not only see in the mouse models but some of your children maybe. When they're old and they have episodes of hypoglycemia the poor PWS pig had more severe and more frequent hypoglycemia. We can treat that and get the blood energy levels back. Then about 13 weeks of age, they did develop what appeared to be hyperphagia and so they went on to a strict maintenance diet. At that point they were at Stewart Laboratory in Missouri. We have now in May shipped them to Pittsburgh and our veterinarians wanted to start them off without having the strict maintenance diet to start. Their weight just suddenly shot up in within a few weeks. We can't say we haven't characterized it in detail. At this point we need more animals so we can actually study the food intake and study the energy levels study; their body composition. The signs are there that we have many of the features. I also point out that our full PWS pig has quite severe scoliosis and a number of your children or young adults may have developed issues with scoliosis. It looks like in the pig that's going to be a good model for the potential bone mineral defects and scoliosis.

Participant:

Larry, I was just wondering how you're getting the autistic diagnosis for the UPD population because you know they're often misdiagnosed and overly diagnosed because of their social functioning? How can you confirm that they are actually autistic and getting into the right population groups?

LR:

That's a good question. We have been stuck with this problem from the start; of using these dental stem cells because we're remotely assessing people. However, there are better and better tools now. The SCQ is what we use. I don’t know if you are familiar with what that is. The SCQ works pretty well. It’s not perfect. If you go into any developmental disability talk where people are using tools to assess ASD, although the gold standard is ADOS-ADI, people use all kinds of other testing tools too. You find that if you ask the question of which tool to use; you get five answers. We had to make a choice and the choice was the SCQ based on the literature on how it's fairly similar in its ability to predict to the ADIR. It’s a much shorter questionnaire to you. We have a good rate of people filling it out appropriately and sending it back. It's a valid point that we may have non-ASD kids in our ASD group. However, from the scores they really split pretty well on that particular test. Now that's all good and well but once we did the molecular work it was more striking because we seem to have picked correctly. If you look at the data, there is a striking difference between the plus and minus group. Yet the minus ASD UPD cases look like the deletion cases for those transcripts. That means that what we're seeing is something unique to that group; that particular group is statistically significant and different from the other three groups that we analyzed; controlled, deletion, and minus ASD for these genes. That was encouraging. That said to us that we probably did something here. We picked something that's different between the two. I agree it'd be great if we could do ADOS-ADI on everybody, but I don't know how many of you had that done with your children. It is not trivial to do the ADOS testing and finding somebody who's qualified to do it, even, is not trivial. I know this because we've had a clinical study on duplication 15q for years and it was probably the most difficult thing to do: to get someone to do formal autism diagnostics. I would love to do that, but I don't think it's practical; especially in terms of trying to collect remotely. It makes it almost impossible. If you have any idea if there's some other test I don't know about? I'm happy to try something else but I think it is working.

Participant:

Hi. Hypothetically, if each of you had a couple of million dollars extra to spend on Prader-Willi research where would you direct the funds?

RW:

There are many expensive aspects of research; particularly if you're including clinical trials in that umbrella. I think that the model that FPWR has now, which is a basement-to-top-floor kind of model (where research or funds are allocated according to scientific merit and feasibility and potential to produce original results that are important in the end) is a very sound strategy across the board.

I think that across the board, everybody could use more funding and more federal government funding in different countries is harder and harder to obtain. Getting seed money from FPWR makes a huge difference and that's why some investigators who've gotten $100,000 from FPWR stay with Prader-Willi syndrome research is because they got their first grant. Because of that first grant they got an NIH grant and then they'll keep working on it. I think actually spreading it thin rather than thinking about one huge investment is actually a really good strategy. It's a it's a well-thought-out strategy. It's not just a default strategy.

LR:

I can send you my address and you can send the check. I agree with what she's saying, and I’ve had interactions with other foundations of this size and it is kind of what I would encourage. In fact, I encourage the Dup15q Alliance to give fellowships to students (to graduate students) because that's four years of commitment on those projects and less money for them actually to commit per year. Actually, in the long term I think it will have a bigger effect of people moving into that field but at the same time, some things are expensive. I'm kind of both ways. I've really been trying to get to the point where we could do single-cell sequencing in our cultures. There's nobody at my university even doing that. It's extremely prohibitive. Given that the structure is in place, if there is a time when you had the standard projects and then you gave away a project that had high merit and that was expensive; I think there's enough good scientists in this particular field that you would probably be able to find one that would be worth spending a little more money on. It cost more but it was the only way to do it right.

RN:

I'll put out another idea; to say I would agree completely with Rachel and Larry. I mean if we each had a couple million dollars could do our work better. We'd have more people in our labs. We’d do it faster because we would have more people who can do work. We need to believe in what we're working on, so we could progress that way. If we had a couple of million each I’d expand out if we had some really wealthy donor come in and say, “I've got 40 million dollars or something like that for Prader-Willi, what would you do?”. What I would want to do is actually create a new Institute where I hand-picked the best Prader-Willi researchers and non Prader-Willi researchers in different fields and bring them all together; put them in one Institute to work together. You know from these types of meetings like FPWR, we get to interact with other investigators and more people; share ideas and start collaborations. If you're in the same place that they're doing that every single day, you're taking a lunch break, you're taking a coffee break, and you're talking ideas.

LR:

I would support that and say that Howard Hughes has recently branched out into this. I don't know if you know about you know HHMI investigators (Howard Hughes Investigators). it's Howard Hughes and this money was left for excellence in science. The way it worked before the strategy they had was they would pick these individuals who they would give enough money to that they didn't have to worry about the money. They've now moved in this structure to a place called Janelia farms and I think it's the first one. I think they're planning another. It's like what Rob is talking about; it’s a more open scientific environment for lots of people who work in slightly different areas to come together, work at a single place, and excel research even further than if we all did it individually. It's kind of an interesting idea that other people have started to do (or at least Howard Hughes has started to do).

TS:

That's all great. We do try. Since we don't have 40 million dollars, I mean to the extent that we can try to bring all of you guys together and encourage collaboration. We as a foundation, we do have different working groups. There's a preclinical animal network that Rachel's involved in. We just had a meeting of a Schaaf-Yang advisory group. We do try to the extent that we can without the 40 million dollars to be doing that and bringing the experts together, encouraging, and putting funds towards collaborations.

LR:

Maybe the family should hear that you guys have done a good job for researchers to hear. The fact that you have two announcements a year is incredible for a foundation. You are funding good research and you're doing it in a structured way that makes sense. You're spending the money the right way.

Participant:

If I understood what you collectively said correctly, it sounds like even though in the mouse models you use reduction of appetite as an outcome. That's not considered to necessarily be equivalent to looking at hyperphagia. My question is in your animal models what criteria would you use to determine whether an animal was experiencing hyperphagia specifically?

RW:

There are animal models that have hyperphagias. There is definition in a rodent with hyperphagia (which is excessive food consumption) for activity levels and they gain weight. There are animals with leptin deficiency, leptin receptor deficiency, POMC deficiency, and all the way up to the appetite pathways. What researchers consider hyperphagia, which is eating too much, but if you're talking about hyperphagia as in thinking about food, asking about food, and obsessing about food; asking when am I going to get seconds? You can't model that in an animal. It’s a very good point: that definition of hyperphagia is difficult to model in any nonverbal animal (which is everybody except us). There are also brain MRI type of imaging protocols that can assess hunger to some degree in animal models. To be honest, animals are not a great model for many psychiatric and behavioral components of human disorders because of that exact problem. We can model some aspects of hyperphagia, food consumption, and patterns of eating; but the thought processes behind that eating behavior are a challenge.

RN:

I think that is an area though that we're going to be able to move into over the next five to ten years with larger animal models; like the pig and other large animal models because the animals are more intelligent. You can do the imaging techniques for the brain, which are also improving all the time and are available; the MRI functional imaging. Although we can't ask an animal what it's thinking or feeling there's a lot of aspects that can be interpreted particularly with the random models where the brain structures are more similar.

RW:

I think that with rodents, they are working for food paradigms, they call them. How many times the animal presses (is willing to press) a lever in order to get a pellet of food? How often do they go back? There are some very sophisticated methods for assessing that.

Participant:

I just wanted to clarify. That was not meant as a criticism. The animal models is more of a curiosity. For example, the advantages of the pig model versus the mouse model and what exactly that would be, but I think you helped answer that with the size of the brain, of the intelligence of the animal, and those types of things.

TS:

Most of the PWS mouse models don't develop the degree of obesity that you might expect. It doesn't mimic the PWS in humans.

RW:

Anybody who's had a labrador retriever or golden retriever knows what hyperphagia looks like. It's like ten seconds; where did it go? When's dinner? You just had dinner, right? So, it is possible to measure those things in animals. In rodents, it's not so easy.

Participant:

Specific question: in the pig model what's the nasal physiology of the pig and in particular the blood-brain barrier in the in the pig compared to the rodents?

RN:

Okay, you got me there. That’s up to a talk with our veterinarian so I don't have the answers for that.

Participant:

Can we talk more about gene activation? It was successful in in a dish, so what are the next steps?

TS:

It was successful in a mouse. Dr. Jang, who's at Duke University screened a library of compounds and found a compound that did activate the maternal allele. He used that (and it's all FPWR funded) in a mouse model of PWS and the mouse model that he used has severe failure to thrive and normally dies. What they saw was an improvement in survival and that the animals that survived also were pretty normal-sized looking. They didn't have that small phenotype or characteristics that you normally see so that's the good news. It seems to work. To do the gene activation, it seems to have a therapeutic effect. The issue with that small molecule is being advanced. Levo Therapeutics is now working with them so it's kind of gone beyond when you start to get into big drug development. That's beyond our budget right now. Levo Therapeutics is now entered into an agreement with them and they're going to do preclinical development of that molecule. They’ll need to do safety testing and then try to move it ahead. The issue with a small molecule approach is that it's not specific for the PWS region. It activates the PWS genes and it activates a bunch of other genes.

From a safety perspective that's going to have to be carefully looked at. What else is it activating and is there any downside to that? We all want new genetic therapies for our kids. If we stay on top of their health, then they're healthy. They can be pretty healthy kids so what we don't want to be doing is giving them some exciting genetic therapy and having some additional problems. It works; and the safety issues will have to be really carefully looked at before we see if it'll go into humans. Then there is the study in a dish which was from a lab and that is a very specific approach. It activates the PWS genes and it doesn't activate other genes. When we talk about delivery, it's a gene-based kind of therapy. You’re getting into how are you going to get that gene into somebody's brain? It’s going to have delivery issues. There are other approaches using CRISPRS to do gene activation and I think the foundation's approach at this point is to try to help push everything forward and answer some of these feasibility questions. We don't know which of those therapies might actually be able to go into people but at this point for me, I don't think we can put all our bets on one approach or another. We'll keep looking at the feasibility to see if a) it will make a difference in kids after they're born, b) if it does, which are the best approaches?, and c) will it work with whatever pharmaceutical company as we can to try to move as many forward?

LR:

I would just add to that that if you follow the parallel in the Angelman field, there was a drug that's a it's called a topoisomerase inhibitor and it worked really well at reactivating ub3 and it worked in the mouse model. It was great. it worked in a dish. I was really good. I think it worked in the mouse. I know we did most of these experiments in the dish and the problem was (and is in all these fields) for all these syndromes—off target effects. The same thing that we were just talking about killed that as a potential drug. You don't want to make things worse. I just want to point out caution. When you get excited about a new compound you really have to be cautious because you don't want to make things worse.

RW:

It’s not a situation that's unique to Prader-Willi syndrome. In cancer therapy this is absolutely classic. All the old drugs were great at stopping the cancer, but they also killed the patient because they work on all cells. Then as soon as people started developing monoclonal antibodies to attack specific receptors that are only present on that particular type of cancer cell, then the patient stays healthy and the tumor is gone. That's an oversimplification but we're aiming for that second line of therapy without having to go through people on the first line. You don't want to do that first line. You just want those those really specific targeted therapies.

LR:

These models that we're talking about up here are really essential at figuring out which of these compounds might be the best. That's going to be the breakthrough as we build better models. You're going to be able to look at the activation at that locus better, right and that's important.

Participant:

I was just wondering if we can tell if you alter the methylation of the maternal genes if they're going to act the same way as the paternal genes? Do we have any idea?

TS:

If you turn on that maternal gene that big transcript turns off UBE3A. In theory, we could cure PWS and give the kids Angelman syndrome which obviously would be a no. The compound that I think was reported by the Duke group, it didn't change the methylation; it changed the histone proteins. They did not see regulation of UBE3A, so it didn't seem to be inducing Angelman syndrome. I don't think that mechanism is all the way worked out.

RN:

I think that was also true with (a researcher’s) result. He was targeting a specific repressor and I don't think that affected right. The other aspect of the ub3 stories though; it's the sort of dogma in the field with UBE3A. I think that's far from proven and we're not in a scientific setting but I could give you scientific reasons why I don't necessarily believe that is correct or proven I think that in terms of therapeutic approaches, that definitely may not be a concern; but if you've changed the methylation of the DNA or the histones and you reactivate, it should have the same effects as if they were expressed with external chromosome. That one concern is what happens with UBE3A, would you affect that or not? That would be the only concern there and then the question is always can you turn it on at the right amount? You don't want to turn it on too superficial levels because that may have effects we can't predict. You want to turn it on just enough to replace the gene functions.

TS:

I guess the encouraging thing from the Duke paper (which wasn't a mouse model) was it did seem to have a positive effect and the animals were healthier after the treatment than before. It was a step forward. Other questions?

Participant:

Why do we have imprinting?

LS:

There is there is a review article theory as to why this is but there's a lot of theories.

RN:

I think they're from a guy named David Haig, an Australian who works at Harvard. He's an evolutionary biologist. Imprinting occurs in mammals. It also occurs in plants and has molecular mechanisms. The world is somewhat different. The idea in mammals is that paternally-expressed genes evolved to make their offspring bigger and stronger and better able to survive, whereas maternal genes evolved to suppress that. This is associated with genes expressed in the embryo and in the placenta. Maternal genes evolved to suppress that, so that every offspring had an equal chance of survival. This is the theory that relates to not humans but other mammals; where they are not monogamous. There'll be many partners. Each male wanted his offspring to succeed and the female wanted all her offspring to have an equal success. This is kind of an arms race between paternal expressed genes and maternal expressed genes. This is a general theory and it that turns out a lot of imprinted genes are growth factors. A lot of paternal express genes improve embryo growth and a lot of maternal genes help suppress that. With something like Prader-Willi, it's not quite so clear because most of what's going on with Prader-Willi is postnatal behavior. There are other imprinted genes that affect maternal behavior postnatally and offspring, so it gets it gets very complicated.

TS:

Yeah it is mom versus dad really. I believe it is time for us to wrap up and so I want to thank our panel.

Susan Hedstrom

Susan Hedstrom is the Executive Director for the Foundation for Prader-Willi Research. Passionate about finding treatments for PWS, Susan joined FPWR in 2009 shortly after her son, Jayden, was diagnosed with Prader-Willi Syndrome. Rather than accepting PWS as it has been defined, Susan has chosen to work with a team of pro-active and tireless individuals to accelerate PWS research in order to change the natural history of PWS. Inspired by her first FPWR conference and the team of researchers that were working to find answers for the syndrome, she hosted her first One SMALL Step walk in 2010 and began the development of the One SMALL Step walk program which now raises over $1.5 million a year for PWS research.

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The mission of FPWR is to eliminate the challenges of Prader-Willi syndrome through the advancement of research. High-quality research will lead to more effective treatments and an eventual cure for this disorder. By working together, we intend to free our loved ones from the burdens of PWS, allowing them to lead full and independent lives.